Optical techniques for imaging membrane domains in live cells (live-cell palm of protein clustering)

It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficult to study by conventional fluorescence microscopy approaches. Photoactivated localization microscopy (PALM) is a super-resolution technique capable of localizing the positions of individual molecules with tens of nanometers precision. Here, we describe a method for imaging membrane proteins using PALM, including live-cell PALM, to detect the molecular clustering of plasma membrane proteins using a statistical cluster-analysis method based on Ripley's K-function. While the method is applicable to a wide variety of proteins in various biological systems, to illustrate the technique, we will image and analyze the clustering behavior of the adaptor protein Linker for activation of T cells (LAT) at the T cell immunological synapse [Williamson, D. J., Owen, D. M., Rossy, J., Magenau, A., Wehrmann, M., Gooding, J. J. , and Gaus, K. (2011). Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol.12, 655-662.].